BES Project No. 27569 EPA Contract No. EP-W-06-026 

Study Title 

Effect of Lindane on the Survival, Development, and Reproduction of the Marine Copepod 

Amphiascus tenuiremis

Block Environmental Services Authors 

David S. Block, Ph.D. 

Nanette Bradbury 

Study Completion Date 

 March 12, 2012 

Submitted To 

RTI International 

3040 East Cornwallis Road 

Research Triangle Park, NC 27709-2194 

Performing Laboratory 

Block Environmental Services, Inc 

2451 Estand Way 

Pleasant Hill, CA 94523 

Laboratory Project ID 

 BES Study No. 27569 

RTI Study No. 0210114.030 

EPA Contract No. EP-W-06-026 
BES Project No. 27569 EPA Contract No. EP-W-06-026

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QUALITY ASSURANCE STATEMENT 

The study conduct, raw data and report for “Effect of Lindane on the Survival and 

Reproduction of the Marine Copepod Amphiascus Tenuiremis” was inspected by the Quality 

Systems Director at Block Environmental Services, Inc (BES) to determine adherence with the 

Quality Assurance Project Plan (QAPP) and laboratory standard operating procedures. Dates of 

study inspections, inspection types, and dates reported to the Program Manager are given below. 

Inspection Date Inspection Type Reported to Program Manager 

 27569 (Lindane)

8/16/2011 in-test, setup 8/16/2011 

9/9/2011 in-test, mating 9/9/2011 

9/26/2011 data, analytical methods verification 9/26/2011 

10/5/2011 data, chemistry 10/10/2011 

12/21/2011 data, biology spreadsheets 12/23/2011 

2/24/2012 draft report 2/27/2012 

3/5/2012 revised draft report 3/6/2012 

BLOCK ENVIRONMENTAL SERVICES, INC. 

________________________________________________ ____March, 9, 2012____

Ronald M. Block, Ph.D. Date 

Quality Systems Director 
BES Project No. 27569 EPA Contract No. EP-W-06-026

KEY STUDY PERSONNEL 

The following BES personnel were responsible for the conduct of the work and reporting of the 

study results. 

David S. Block, Ph.D. 

3/12/2012 

Program Manger/Technical Report Writer Date 

Ronald M. Block, Ph.D Quality Systems Director 

Nanette Bradbury Senior Biologist/Technical Report Writer 

Terence Clohesy Biologist 

Sebastian Castro Biologist 

Amy Ayala Biologist 

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BES Project No. 27569 EPA Contract No. EP-W-06-026

DATA RETENTION 

All records that would be required to reconstruct the study will be maintained. The original raw 

data and the original final report will be maintained indefinitely in the form of a hard copy to 

comply with EPA record keeping regulations. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

TABLE OF CONTENTS 

QUALITY ASSURANCE STATEMENT .................................................................................. 2

KEY STUDY PERSONNEL........................................................................................................ 3

DATA RETENTION .................................................................................................................... 4

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TABLE OF CONTENTS ............................................................................................................

ABSTRACT..................................................................................................................................

1 INTRODUCTION..................................................................................................................

2 MATERIALS AND METHODS ........................................................................................

2.1 QAPP/Protocol............................................................................................................. 

2.2 Culturing test organisms.............................................................................................. 

2.2.1 Source of copepods.................................................................................................. 

2.2.2 Culture medium & diet ............................................................................................ 

2.2.3 Laboratory Reagent Water for Culture Media Preparation...................................... 

2.2.4 Culture Sediment ..................................................................................................... 

2.2.5 Photoperiod.............................................................................................................. 

2.3 Toxicity test procedure ................................................................................................ 

2.3.1 Test chemical ........................................................................................................... 

2.3.2 Preparation of test solutions..................................................................................... 

2.3.3 Test equipment......................................................................................................... 

2.3.4 Collection of copepod nauplii in preparation for the test ........................................ 

2.3.5 Diet during testing.................................................................................................... 

2.3.6 Daily observations ................................................................................................... 

2.3.7 Test conditions......................................................................................................... 

2.3.8 Lindane concentrations in test solutions.................................................................. 

2.3.9 Statistical analyses................................................................................................... 

3 RESULTS .............................................................................................................................

3.1 Physico-chemical properties of test solutions.............................................................. 

3.2 Measured lindane concentrations in test solutions....................................................... 

3.3 Biological data ............................................................................................................. 

3.3.1 Survival on day 16 ................................................................................................... 

3.3.2 Survival on day 36 ................................................................................................... 

3.3.3 Day 16 Versus Day 36 Survival............................................................................... 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

3.3.4 Development – Maturation time between naupliar and copepodite stages.............. . 17

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3.3.5 Development – Maturation time between copepodite and adult stages................... 

3.3.6 Development – Maturation time between naupliar and adult stages....................... 

3.3.7 Sex ratio ................................................................................................................... 

3.3.8 Reproduction – time to first egg extrusion .............................................................. 

3.3.9 Reproduction – number of hatched nauplii from first brood ................................... 

3.3.10 Reproduction – time to second egg extrusion...................................................... 

3.3.11 Reproduction – number of hatched nauplii from second brood .......................... 

3.3.12 Reproduction – Total number of nauplii per two brood females......................... 

3.3.13 Reproduction - fertilization success..................................................................... 

3.4 Summary of NOEC and LOEC for all endpoints........................................................ 

4 DISCUSSION.......................................................................................................................

4.1 Quality Objectives and criteria .................................................................................... 

4.2 discussion of results..................................................................................................... 

5 ACKNOWLEDGEMENTS ................................................................................................

6 REFERENCES.....................................................................................................................

7 FIGURES..............................................................................................................................

Figure 1: Layout of Microplate Test Chamber ................................................................ 

8 TABLES.................................................................................................................................

Table 1: Method for preparation of lindane primary stock solution................................ 

Table 2: Method for preparation of lindane test solutions............................................... 

Table 3: Water quality parameters: pH, salinity, dissolved oxygen ................................ 

Table 4: Water quality parameter: daily temperature ...................................................... 

Table 5: Summary of water quality parameters............................................................... 

Table 6: Measured lindane test concentrations................................................................ 

Table 7: Summary of measured lindane test concentration............................................. 

Table 8: Survival on day 16............................................................................................. 

Table 9: Survival on day 36............................................................................................. 

Table 10: Time to transformation to copepodite stage .................................................... 

Table 11: Time in copepodite stage................................................................................. 

Table 12: Time to transformation to adult stage.............................................................. 

Table 13: Sex ratio........................................................................................................... 

Table 14: Time to first egg extrusion since date of pairing ............................................. 

Table 15: Number of nauplii hatched from first brood.................................................... 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 16: Time to second brood since date of pairing..................................................... . 35

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Table 17: Number of nauplii hatched from second brood ............................................... 

Table 18: Total number of nauplii that hatched from two successive broods................. 

Table 19: Fertilization success......................................................................................... 

Table 20: Summary table of NOEC and LOEC for all endpoints................................... 

9 APPENDIX I: QUALITY ASSURANCE PROJECT PLAN (QAPP)............................

10 APPENDIX II: QUALITY OF LABORATORY WATER AT BES ..............................

11 APPENDIX III: LINDANE CHEMISTRY DATA REPORT.........................................

12 APPENDIX IV: STUDY PROTOCOL (P27569)............................................................

13 APPENDIX V: DEVIATIONS FROM STUDY PROTOCOL......................................

13.1 List of deviations........................................................................................................ 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

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ABSTRACT 

The current study assessed the chronic sensitivity of the meiobenthic copepod Amphiascus 

tenuiremis to lindane concentrations of 2.5, 5, 10, 15, and 20-μg/L using a microplate-based lifecycle screening assay. Copepod survival in the laboratory control and carrier control treatments 

on Day 16 was 82% and 79%, respectively with survival in each control treatment decreasing to 

77% by Day 36. The 15 and 20 μg/L lindane concentrations significantly affected copepod 

survival on both Day 16 and 36 time points relative to the control response. None of the lower 

lindane concentrations significantly affected copepod survival. Lindane had a significant 

inhibitory effect on A. tenuiremis development into both copepodite and adult life-stages. 

Specifically, lindane significantly increased the time to develop from the naupliar to the 

copepodite life-stages and also significantly increased the time necessary for exposed copepods 

to mature into sexually mature adults. Based on the development data obtained during the study, 

NOEC and LOEC values were calculated to be 2.5μg/L and 5μg/L respectively. None of the 

lindane concentrations significantly affected the ratio of male:female copepod development. 

Lindane was also observed to have a significant effect on copepod reproduction, with a reduction 

in the percentage of successful mating pairs and a reduction in the total number of viable eggs 

and hatched nauplii. Based on the mating success data, NOEC and LOEC values were calculated 

to be 5μg/L and 10μg/L, respectively. Based on the total offspring production data, the NOEC 

and LOEC were calculated to be 10 μg/L and 15 μg/L, respectively. Time-weighted measured 

lindane concentrations were within 20% of the nominal values. All statistical analyses were 

performed using measured values. 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

1 INTRODUCTION 

The U.S. Environmental Protection Agency (EPA) is currently implementing the Endocrine 

Disruptor Screening Program (EDSP). To support this program, EPA has contracted with RTI 

International (RTI) to provide comprehensive toxicological testing services. These services were 

designed to assist the EPA in validating a suite of longer term Tier 2 in vivo, arthropod 

ecotoxicological tests for identifying and characterizing endocrine effects through exposure to 

pesticides, industrial chemicals, and environmental contaminants. The objective of the validation 

studies are two-fold: one, ascertain the value-added from a mysid two-generation growth and 

reproduction test as compared to existing mysid life cycle methodologies; and two, evaluate a 

microplate-based copepod development and reproduction test method using the marine 

harpacticoid copepod Amphiascus tenuiremis. In addition to method validation, the results and 

conclusions from these studies will be used to develop appropriate guidance documents for peer 

review of the methods and standardize technical guidance and test guidelines in support of the 

Office of Prevention, Pesticides and Toxic Substances regulatory programs. These studies were 

conducted under the study protocol, applicable Quality Assurance Project Plans (QAPPs), 

relevant program and facility standard operating procedures (SOPs), and published guidance 

documents. 

The purpose of work to be conducted by Block Environmental Services, Inc. (BES) under this 

assignment is to demonstrate a copepod development and reproduction test based on ASTM E 

2317-04 “Standard guide for conducting renewal microplate-based life-cycle toxicity tests with a 

marine meiobenthic copepod” and the June 2009 proposed OECD Test Guidelines for the 

Harpacticoid Copepod Development and Reproduction Test with Amphiascus tenuiremis (OECD 

2009). As indicated above, the test is intended to serve as a higher tier test with an aquatic 

arthropod for collecting definitive concentration-response information on adverse effects suitable 

for use in ecological risk assessment. Specifically, the design enables the collection of biological 

data from this copepod species in order to facilitate the measurement of development, 

reproductive and life-table parameters, which provide sensitive and ecologically relevant 

endpoints that can be used to determine the potential chronic toxicity of many water-borne 

contaminants. 

Block Environmental Services (BES) conducted one copepod development and reproduction test 

with lindane using the proposed OECD (OECD, 2009) and published ASTM test guidelines 

(ASTM, 2004) Endpoints ranged from survival, development, and specific reproductive output 

measures, including time to first and second brood, number of offspring, reproductive success. 

The experimental phase of the study was conducted from August 16, 2011 to September 21, 

2011. The original raw data, protocol, and technical report produced during this study are stored 

at the BES laboratory, located in Pleasant Hill, California. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

2 MATERIALS AND METHODS 

2.1 QAPP/PROTOCOL 

This study was conducted according to the procedures outlined in the Quality Assurance Project 

Plan (QAPP) for Task Order 30 Mysid/Copepod Inter laboratory Test Evaluation. The QAPP can 

be found in Appendix I. The study protocol was designed to assess the effect of lindane on the 

survival rate, development, mating success and reproductive output of the copepod A. 

Tenuiremis (OECD, 2009). Where deviations occurred they have been listed in and discussed 

where required in Appendix VI. 

2.2 CULTURING TEST ORGANISMS 

2.2.1 Source of copepods 

A gift of A. tenuiremis was received from Professor G.T. Chandler (University of South 

Carolina, School of Public Health, Columbia, South Carolina, USA) on March 19, 2011. 

Information on the original source of this copepod population can be found in ASTM (2004). 

On arrival at the BES Laboratory, copepod cultures were maintained in food grade polyethylene 

(Tupperware®) containers containing approximately 2 liters of culture media and one centimeter 

of 63μm sieved and washed sediment. 

2.2.2 Culture medium & diet 

Care for the copepod culture at BES followed the instructions presented by the Chandler 

Laboratory (Columbia, South Carolina). Synthetic Instant Ocean® seawater was used to prepare 

the copepod culture medium. The synthetic seawater was prepared by dissolving Instant Ocean®

sea salt into laboratory grade deionized water in accordance with the manufacturer’s instructions 

at appropriate amounts and volumes necessary to achieve 30 ‰ salinity. Copepod culture media 

was prepared by filtering the synthetic seawater through a 0.2μm hydrophilic polyethersulfone 

membrane (VWR® 47mm glass filter holder; Pall Corporation, Port Washington, USA). The 

quality of the culture media was checked weekly using measurements of dissolved oxygen, pH, 

salinity, and temperature. The culture media was siphoned and replenished two times per week. 

During culture media renewal, the fresh media was amended on an alternating basis with either 

the live alga Dunaliella tertiolecta, a chlorophyte, or Isochrysis galbana, a chrysophyte. The 

algae were added to the copepod culture medium to provide a final density of approximately 

2.8x108 algal cells/L. 

2.2.3 Laboratory Reagent Water for Culture Media Preparation 

Laboratory reagent water is prepared by processing carbon-scrubbed municipal water in tandem 

through a reverse osmosis membrane and deionization resin. The quality of the laboratory water 

is checked once annually, at a minimum. The analytical report for the most recent samples, 

which were collected and analyzed in May 2011, is provided in Appendix II. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

2.2.4 Culture Sediment 

Concentrated sediment previously filtered through a 63μm wire mesh sieve was received as a 

gift from Professor G.T. Chandler and stored in the dark at 4°C prior to use. According to the 

Chandler Laboratory, the sediment was obtained from Bread and Butter Creek, located near 

Charleston, South Carolina. The sediment was prepared in accordance with the Chandler 

Laboratory procedures. Specifically, a tablespoon of concentrated sediment was resuspended in 

one liter of 0.2μm 30 ‰ synthetic seawater using an electric blender (Waring Products, 

Torrington, CT). The sediment slurry was allowed to settle overnight and then decanted. The 

resuspension and settling process was immediately repeated. The final washed sediment was 

added to the copepod cultures when the quantity of in-culture sediment had diminished so that 

the bottom of the polyethylene container was visible. 

2.2.5 Photoperiod 

A 12-h light (min-max: 4,090-4,520 Lux) and 12-h dark photoperiod was maintained throughout 

the culturing of A. tenuiremis. 

2.3 TOXICITY TEST PROCEDURE 

2.3.1 Test chemical 

Three samples of the test chemical were received from the EDSP Chemical Repository, housed 

at Battelle's Pacific Northwest Division, Sequim, Washington. Upon receipt at BES, the test 

substances were stored at room temperature in the original container in a dark, ventilated cabinet. 

Concentrations were adjusted for the purity of the test substance and are presented as active 

ingredient (a.i.). The following information was provided: 

Name: Lindane

CAS registry number: 58-89-9 

Source: Battelle Marine Science Laboratory, Sequim 

WA 98382, U.S.A. 

Lot / Batch number: Lot: 11419EB

Purity: >99.6%

Form White powder 

Date of receipt at BES March 11, 2011 

Location of storage Fume hood in chemical storage room 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

2.3.2 Preparation of test solutions 

The primary stock solution of lindane was prepared only once on 7/16/2011 and stored in an 

amber glass bottle at 4°C in Sample Locker 1. This primary stock solution was used throughout 

the remainder of the 36-day test. Lindane was weighed using an analytical balance (Sartorius 

Series A 200S) and dissolved into pesticide grade acetone to achieve a stock concentration of 

80μg/ml as detailed in Table 1. The primary stock concentration was selected so that the total 

acetone carrier volume would not exceed 0.05% in any test treatment. Acetone was stored at 

room temperature in a self-closing organic solvent safety cabinet. Acetone was measured using a 

glass graduated cylinder. 

Nominal lindane test concentrations of 2.5, 5, 10, 15, and 20 μg/L were prepared from the 

lindane primary stock as detailed in Table 2. Test solutions were prepared every 3 days, on the 

day of water change. Calculated amounts of the primary stock solution was spiked into 500 mL 

glass volumetric flasks containing 0.2μm 30 ‰ synthetic Instant Ocean® seawater (see 2.2.2 and 

2.2.3) using Hamilton glass volumetric syringes with stainless-steel needles. Test solutions were 

not prepared by serial dilution, but instead prepared independently by pipetting the calculated 

amount of primary stock solution directly into the designated volumetric flask. To prevent bias, 

the acetone solvent carrier was added to an aliquot of the control solution in a volume equivalent 

to the highest amount added into the lindane treatments. Once prepared, test solutions were 

sealed, mixed for 1-hr using Teflon® coated stir-bars, and stored in the chemical storage room 

until used. 

2.3.3 Test equipment 

The 36-day microplate test design followed the draft OECD (2009) guideline. The following 

equipment was used: 

 96-well polystyrene microplates (Corning Costar 3474, Lot number 16011004, exp 

6/18/2014); 

 12-well polystyrene microplates (Corning Costar 3477 Netwell® (purchased from VWR, 

USA) with a 74μm polyester mesh that retains gravid females but allows nauplii through 

the mesh to the bottom of the well; 

 Oxygen meter suitable to measure >60mL volumes (YSI 550A); 

 pH meter (Orion 230A); 

 Conductivity meter for salinity measurements (YSI 30); 

 Temperature recorder measuring continuous air temperature within environmental 

chamber (Dixon DT8 Recorder #39585); 

 Alcohol thermometer immersed in a glass beaker containing dilution water located in the 

humidity chamber. Thermometer is calibrated to an NIST certified thermometer every 

three months; 

 Two inverted stereo compound microscopes with dark-field (Two AmScope IN200B 

with No equipment ID); 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

 Two dissection stereomicroscope, with dark-field illumination and LED episcopic 

illumination (Two AmScope SM-1TZ-PL); 

 Temperature and light regulated environmental chamber (Lab-Line Environ-Shaker 

#3597 designated as environmental chamber #3, located in Testing Room #4); 

 One 0.047 m3 plastic container with sealable lid to create humidity chambers for the 

microplates (to minimize evaporative loss of test solutions). A plexiglass platform 

(perforated with 0.3cm3 holes) was supported above standing water inside the sealed 

plastic container; 

 Stainless-steel mesh sieves (0.090mm and 0.106mm) to culture, capture and sort 

copepods; 

 Glass Pasteur pipettes silanized with Enviro-Coat, OmniPur*, Non-hazardous silane 

replacement to prevent sticking of copepods onto the glass surface. Enviro-Coat (EM4190) was purchased through VWR; 

 Adjustable single channel volumetric pipettes (5-50μL, 100-1000μL) for inoculating 

algae cultures used as food for cultures and tests 

 Adjustable multi-channel volumetric pipette (1-10 μL) for adding food and for adding 

primary stock solution during the preparation of test solutions; 

 10, 50, and 250 μL Hamilton glass volumetric syringes with beveled stainless-steel 

needles for microplate water changes and test solution preparation; 

 Haemacytometer cell counting chamber (Neubauer depth of chamber 0.1mm) to count 

algae during food preparation; 

 47mm Glass Filter Holder (VWR USA, Port Washington, USA); 

 0.2 μm sterile polyethersulfone membrane, 47 mm (Pall, Port Washington, USA) 

2.3.4 Collection of copepod nauplii in preparation for the test 

Approximately 300 gravid female copepods were collected from laboratory stock mud cultures 

by sieving across 106 μm steel mesh. Gravid females retained on the 106 μm sieve were rapidly 

rinsed with 30 ‰ dilution water into polystyrene Petri dishes. The organisms were transferred 

using enviro-coat treated glass transfer pipettes into wells of 12-well Netwell® microplates filled 

with 0.2μm filtered seawater. Each well of the Netwell® microplate is equipped with a 74 μm 

polycarbonate mesh filter (Corning Costar 3477), suspended 4 mm above the bottom of the 

microplate. The 74 μm polyester mesh retains gravid female copepods but allows hatched 

nauplii to fall through to the bottom of the well. Each well contained approximately 50-60 

gravid females. 

Newly hatched nauplii (≤24-h) that had migrated through the mesh filter were collected, pooled, 

and haphazardly allocated to individual microwells of ultra-low attachment Hydrogel® coated 

(i.e., hydrophilic) polystyrene 96-well microplates (Corning Costar 3474) containing 250 μL of 

each test solution. For each lindane test concentration and control, a total of 90 individuals were 

allocated across three 96-well plates. Each 96-well plate had 30 individuals placed into columns 

2 through 11 of rows B, D and F (column 1 and 12 were purposely left empty). See Figure 1

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

The 96-well plates were randomly assigned a concentration using a random number table. All 

subsequent handling, loading, observations and feeding were done without respecting any order 

to ensure that handling bias would not be construed as being a chemical-dependent effect. For 

example, if experimental units are handled in treatment or concentration order, then some timerelated effect, such as operator fatigue or other error, could lead to greater or lesser effects at the 

higher or lower concentrations. Only one test chemical concentration was tested per microplate 

to reduce chances for microwell cross contamination. 

2.3.5 Diet during testing 

During the chemical exposure period, copepods were fed every six days. Individual copepods in 

test chambers were fed 2μL of a mixed-algal cell suspension (centrifuged, pelletised and reconstituted in fresh 30 ‰ seawater to 107 cells/mL) at a ratio of 1:1:1 Dunaliella tertiolecta, 

Isochrysis galbana and Phaeodactylum tricornutum. The algal cell density added to each test 

microwell at each feeding was 2 x 104 cells/microwell. 

2.3.6 Daily observations 

Nauplii were monitored daily via inverted stereomicroscope for development into the copepodite 

juvenile stage. Once becoming copepodites, exposed A. tenuiremis were then monitored for the 

development of secondary sexual characteristics and time, in days, to reproductive adult. Upon 

reaching adulthood, individual virgin female and male copepods were randomly mated (pairwise 

within each treatment) in fresh microwells (wells C and E) using the exposure parameters and 

conditions described above. Mating pairs were monitored daily for reproductive success (egg 

extrusion and fertility) and numbers of offspring successfully hatching for each pair. 

2.3.7 Test conditions 

Test conditions are summarized in Table 3. Essential physico-chemical parameters of the 

dilution water used to prepare the test solutions were measured on each day a water change took 

place. Aliquots of the dilution water were analyzed for conductivity/salinity, pH, and dissolved 

oxygen on YSI 30, Orion 230A, and YSI 550A handheld meters, respectively. Test vessels were 

stored in a temperature and light controlled environmental chamber set at 25°C with a light 

intensity ranging from 4,090 to 4,520 Lux. Due to small volumes within the microwells, direct 

temperature was not measured in the test vessel solutions. Temperature was recorded twice daily 

from a calibrated alcohol thermometer immersed in a bottle containing dilution water placed near 

test vessels. The thermometer is calibrated quarterly against an NIST traceable thermometer. 

Physico-chemical parameters were not measured in freshly made test solutions (referred to here 

as ‘Ups’) and test solutions drawn out of test vessels (i.e. ‘Downs’) in order to prevent lindane 

cross contamination to other ongoing toxicity studies. 

2.3.8 Lindane concentrations in test solutions 

Samples for determining lindane concentration were collected from freshly prepared test 

solutions in the case of Up samples and from pooled solutions from test vessels in the case of 

Down samples. Samples were collected and sealed into 125 ml amber glass bottles stored in 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

Sample Locker 1 at 4°C. Samples were sent throughout the study to North Coast Laboratories, 

LTD. (Arcata, California, USA; CA-ELAP #1247) in an ice chest cooled with packs of Blue 

Ice® (Rubbermaid). Chemical analysis of the test solutions were performed by gas 

chromatography – electron capture detection (GC-ECD) using EPA method 505. Quantities 

were determined by correlating signal responses against reference calibration standards of known 

lindane concentrations prepared in hexane. Results were submitted in audited reports 1109247 

and 1109490 (Appendix III). 

2.3.9 Statistical analyses 

Mean and standard deviations (SD) were calculated using MicrosoftTM Excel 2000. Survival, 

development, and reproduction data were analyzed using SigmaStat version 2.03 for Windows 

(IBM, Inc. (formerly SPSS, Inc.), Armonk, New York, USA). All statistical conclusions were 

made at the 95% level of certainty except in the cases of Levene’s Test and Shapiro-Wilk's Test, 

in which the 99% level of certainty was applied. The following procedures were used: 

 Data for each endpoint were analyzed using Levene's Test (USEPA, 2002) as a check on 

the assumption of homogeneity of variance implicit in parametric statistics. 

 Shapiro-Wilk's Test for normality (USEPA, 2002) was conducted in order to compare the 

observed sample distribution with a normal distribution. The assumption that 

observations are normally distributed must be validated before subsequent analyses 

following parametric procedures, can be performed. If the data were not normally 

distributed, then a non-parametric procedure was used for subsequent analyses. 

 Prior to analysis for statistically significant effects, an analysis of variance (ANOVA) 

was conducted on each data set (except survival and sex ratio data) to determine if a 

treatment group in the data set was different from the other groups in that data set. 

 Post-hoc pair wise comparisons between the solvent control and each treatment level, 

including the dilution water control, were performed using Dunnett's Test (Dunnett, 

1955, 1964), which is a parametric procedure. If necessary, mean values were 

transformed by using square root, arcsine square root, or log conversion procedures. The 

multiple comparison adjustment was utilized to determine the NOEC and LOEC for each 

test endpoint. The Bonferroni pairwise mutlitple comparison procedure was utilized to 

obtain P values between the carrier control and treatement groups since the SigmaStat 

program does not calculate individual pairwise P values in the Dunnett’s test. 

 All proportional data points such as survival rates and sex ratios were analyzed using 

Pearson’s chi- square goodness-of-fit test (R X C contingency tables) (Piegorsch and 

Bailer,1997). 

For clarity, the definition for NOEC and LOEC are described below: 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

 No-Observed-Effect Concentration (NOEC) is the concentration of lindane tested which 

has no effect on the organism in the test, according to the statistical test used for analysis. 

 Lowest-Observed-Effect Concentration (LOEC) is the lowest tested concentration of 

lindane which has an effect on the organism in the test that is different from the control 

(or solvent control), according to the statistical test used for analysis. 

3 RESULTS 

3.1 PHYSICO-CHEMICAL PROPERTIES OF TEST SOLUTIONS 

The baseline physico-chemical measurements for pH, salinity and dissolved oxygen are 

presented in Table 3. Daily temperature measurements from the environmental chamber dilution 

water vessel are presented in Table 4. Table 5 provides an overall summary of these 

measurements and shows that temperature was 25.1 ± 0.6°C, pH was within 8.2 ± 0.1, salinity 

was within 29.7 ± 0.6‰, and dissolved oxygen was 97.4 ± 5.3 of air saturated value. In terms of 

physico-chemical properties, recommendations listed in the QAPP for this study (Appendix I 

Table 3) were met. 

3.2 MEASURED LINDANE CONCENTRATIONS IN TEST SOLUTIONS 

Test concentrations of lindane were reported by North Coast Laboratories LTD, Arcata, 

California, USA. A copy of the audited reports numbered 1109247 and 1109490 is presented in 

Appendix III. Concentration data, including Quality Assurance/Quality Control spikes, are 

reported in Table 6. Time weighted values of the measured lindane concentrations and percent 

of nominal calculations are summarized in Table 7. Time weighted values were generated by 

calculating the arithmetic mean between all Up and Down samples throughout the 36-day test 

period for each treatment level. These tables indicate concentrations of the test substance ranged 

from 75 to 87 percent of the nominal concentration, which is within the criteria specified in 

Appendix I. In this report, all results are expressed in term of nominal values. 

3.3 BIOLOGICAL DATA 

A summary of the statistical analyses performed on the biological observations is presented in 

Section 2.3.9. Reports from all statistical analysis are also provided in Appendix IV. 

3.3.1 Survival on day 16 

Copepod survival data on Day 16 of the study are presented in Table 8. Treatment means, 

including the dilution water control, were statistically compared to the carrier control using 

Pearson’s chi- square goodness-of-fit test. In summary, mean survival in the control and the 

carrier control group was 82% and 79%, respectively. A statistical difference between the 

control means was not evident (P=0.706). Survival in the test treatments ranged from 63 to 79%. 

Copepod survival between the carrier control and 20 ug/L lindane treatment was statistically 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

reduced (P=0.033). No significant effects relative to the carrier control group were observed in 

the 2.5 (P=0.855), 5 (P=0.175), 10 (P=0.597), and 15μg/L (P=0.067) test treatments. 

3.3.2 Survival on day 36 

Recording survival observations of individual copepods was concluded upon test termination on 

day 36. Treatment means, including the dilution water control, were statistically compared to the 

carrier control using Pearson’s chi- square goodness-of-fit test. On Day 36, the control and 

carrier control group each had a mean survival rate of 77%(P=0.860). Table 9 lists the 

percentage survival for all groups. There was a statistically significant difference for survival 

(p<0.05) for the 15 (P=0.017) and 20μg/L (P=0.025) concentrations relative to the carrier control 

group. No significant effects relative to the carrier control group were observed in the 2.5 

(P=0.862), 5 (P=1.0), and 10μg/L(P=0.497) test treatments. The survival result in the 5μg/L test 

concentration reflects data from only two replicates; replicate 1 was accidentally dropped by an 

operating technician on day 25. 

3.3.3 Day 16 Versus Day 36 Survival 

Day 16 and Day 36 mean survival rates were statistically compared within treatment using 

Pearson’s chi- square goodness-of-fit test. No statistically significant differences (p>0.05) in 

survival was observed within control (P=0.461), carrier control (P=0.858), 2.5 (P=0.597), 5 

(P=0.405), 10 (P=0.738), 15 (P=0.442), and 20 μg/L (P=0.759) lindane treatment groups 

between the test midpoint and test termination. 

3.3.4 Development – Maturation time between naupliar and copepodite stages 

Copepod development from the nauplius to copepodite stages is presented in Table 10. 

Treatment means, including the dilution water control, were statistically compared to the carrier 

control using a One Way ANOVA test in conjunction with Dunnett’s post-hoc multiple 

comparison procedure. In summary, mean development time in the control and the carrier 

control group was 9.5 and 9.3 days, respectively. A statistical difference between the control 

means was not evident (P=0.593). There was a statistically significant (p<0.05) increase in the 

number of days for nauplii to maturate into the copepodite stage for lindane concentrations of 5 

(P=0.008), 10 (P=0.001), 15 (P=0.001), and 20 μg/L (P=0.001). No significant effect relative to 

the carrier control group was observed in the 2.5μg/L (P=1.0) test treatment. Based on these 

statistical results, NOEC and LOEC values were determined to be 2.5 and 5μg/L, respectively. 

3.3.5 Development – Maturation time between copepodite and adult stages 

Copepod development from the copepodite stage into virgin adults is presented in Table 11. 

Treatment means, including the dilution water control, were statistically compared to the carrier 

control using a One Way ANOVA test. In summary, mean development time in the control and 

the carrier control group was 8.1 and 8.2 days, respectively. A statistical difference between the 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

control means was not evident (P=0.725).). No statistically significant effects (P=0.1) relative to 

the carrier control group were observed in the 2.5, 5, 10, 15and 20μg/L test treatments. Based on 

these statistical observations, NOEC and LOEC values were determined to be 20 and >20μg/L, 

respectively. 

3.3.6 Development – Maturation time between naupliar and adult stages 

Copepod development from the nauplius to adult stages is presented in Table 12. Treatment 

means, including the dilution water control, were statistically compared to the carrier control 

using a One Way ANOVA test in conjunction with Dunnett’s post-hoc multiple comparison 

procedure. In summary, mean development time in the control and the carrier control group was 

17.4 and 17.5 days, respectively. A statistical difference between the control means was not 

evident (P=0.856). There was a statistically significant (p<0.05) increase in the number of days 

from nauplius to virgin adult for lindane concentrations of 5 (P=0.032), 10 (P=0.001), 15 

(P=0.001), and 20 μg/L (P=0.001). No significant effect relative to the carrier control group was 

observed in the 2.5μg/L (P=1.0) test treatment. Based on these statistical results, NOEC and 

LOEC values were determined to be 2.5 and 5μg/L, respectively. 

3.3.7 Sex ratio 

The proportion of females to males is presented in Table 13. Treatment means, including the 

dilution water control, were statistically compared to the carrier control using Pearson’s chisquare goodness-of-fit test. The average gender ratio (female to male) in the control and carrier 

control groups was 1.4 and 1.2, respectively. There was not a statistically significant difference 

(p>0.05) between the control means (P=0.763). No significant effect relative to the carrier 

control group was observed in the 2.5 (P=0.337), 5 (P=0.567), 10 (P=0.900), 15 (P=0.909) and 

20μg/L (P=0.209) test treatments. The sex ratios observed in the control and carrier control 

treatments were within the Quality Criteria specified in Appendix I, Table 3, although the 

standard deviations exceeded ± 0.2 (± 0.4 and ± 0.5, respectively). 

3.3.8 Reproduction – time to first egg extrusion 

The average time to first egg extrusion in females following the mating of virgin male and 

female copepods is presented in Table 14. Treatment means, including the dilution water control, 

were statistically compared to the carrier control using a One Way ANOVA test. The average 

time to first egg extrusion in the control and carrier control groups was 2.4 and 2.3 days, 

respectively). There was not a statistically significant difference (p>0.05) between the control 

means (P=0.656). No statistically significant differences in the time to egg extrusion were 

observed among the test treatments when compared to the carrier control group (P=0.396). It 

should be noted that no mated females became gravid in replicate 2 of both the 15 and 20μg/L 

test treatments. Organisms were observed for a minimum of six days and a maximum of twelve 

days following mating. The time to first clutch observed in the control and carrier control 

treatments were within the Quality Criteria specified in Appendix I, Table 3. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

3.3.9 Reproduction – number of hatched nauplii from first brood 

The average number of hatched nauplii from the first egg extrusion following the mating of 

virgin male and female copepods is presented in Table 15. Treatment means, including the 

dilution water control, were statistically compared to the carrier control using a One Way 

ANOVA test in conjunction with Dunnett’s post-hoc multiple comparison procedure. The 

average number of first brood nauplii in the control and carrier control groups was 5.9 and 5.8 

nauplii per female, respectively). There was not a statistically significant difference (p>0.05) 

between the control means (P=0.970). There was a statistically significant (P=0.031) decrease in 

first brood offspring in the 20μg/L (P=0.022) test treatment when compared to the carrier 

control. No statistically significant effects relative to the carrier control group were observed in 

the 2.5 (P=1.0), 5 (P=1.0), 10 (P=1.0), and 15μg/L (P=0.459) lindane concentrations. However, 

a large standard deviation in the 15μg/L treatment may have precluded a calculated statistical 

difference since the offspring produced were 39% of the offspring measured in the carrier control 

group. 

3.3.10 Reproduction – time to second egg extrusion 

The average time to second egg extrusion in female copepods is presented in Table 16. 

Treatment means, including the dilution water control, were statistically compared to the carrier 

control using a One Way ANOVA test. The average time to second egg extrusion in the control 

and carrier control groups was 2.2 and 2.3 days, respectively. There was not a statistically 

significant difference (p>0.05) between the control means (P=0.709). No statistically significant

differences in the time to egg extrusion were observed among the test treatments when compared 

to the carrier control group (P=0.517). The 20μg/L test treatment data were excluded from 

statistical analysis since mated females did not produce a second brood in any of the treatment 

replicates. 

3.3.11 Reproduction – number of hatched nauplii from second brood 

The average number of hatched nauplii from the second egg extrusion following the mating of 

male and female copepods is presented in Table 17. Treatment means, including the dilution 

water control, were statistically compared to the carrier control using a One Way ANOVA test in 

conjunction with Dunnett’s post-hoc multiple comparison procedure. The average number of 

second brood nauplii in the control and carrier control groups was 6.0 and 6.7 nauplii per female, 

respectively. There was not a statistically significant difference (p>0.05) between the control 

means (P=0.420). There was a statistically significant (p<0.05) decrease in second brood 

offspring in the 15 (P=0.002) and 20μg/L (P=0.002) test treatments when compared to the carrier 

control. No statistically significant effects relative to the carrier control group were observed in 

the 2.5 (P=1.0), 5 (P=1.0), and 10μg/L (P=1.0) lindane concentrations 

3.3.12 Reproduction – Total number of nauplii per two brood females 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

The average number of nauplii per female from mating pairs with two broods is presented in 

Table 18. Treatment means, including the dilution water control, were statistically compared to 

the carrier control using a One Way ANOVA test in conjunction with Dunnett’s post-hoc 

multiple comparison procedure. The total number of offspring/female in the control and carrier 

control groups was 11.7 and 12.5, respectively. There was not a statistically significant 

difference (p>0.05) between the control means (P=0.625). There was a statistically significant

(p<0.05) decrease in nauplii per female in the 15 (P=0.033) and 20μg/L (P=0.007) test treatment 

when compared to the carrier control. No statistically significant effects relative to the carrier 

control group were observed in the 2.5 (P=0.468), 5 (P=1.0), and 10μg/L (P=1.0), lindane 

concentrations. The total number of viable offspring produced through two clutches in the 

control and carrier control treatments were within the Quality Criteria specified in Appendix I, 

Table 3. Based on these statistical results, NOEC and LOEC values were determined to be 10 

and 15μg/L, respectively. 

3.3.13 Reproduction - fertilization success 

Fertilization success is defined as when mated gravid females extrude two successive clutches 

that produce viable offspring. Treatment means, including the dilution water control, were 

statistically compared to the carrier control using t-tests. The average fertilization success rates in 

the control and carrier control treatments were 85 and 59%, respectively (Table 19). While the 

fertilization rate in the carrier control group was reduced by 26% relative to the control group, 

the difference was not statistically significant (P=0.102). Nevertheless, the reduced rate may be 

indicative of a solvent effect in this particular study. There was a statistically significant 

(p<0.05) decrease in the fertilization success rate in the 10 (P<0.001), 15 (P<0.001) and 20μg/L 

(P<0.001) test treatments relative to the carrier control. No significant effects relative to the 

control group were observed in the 2.5 (P=0.8) and 5μg/L (P=0.583) lindane concentrations. 

Gravid female copepods that did not hatch their second brood hatch by Day 36 were excluded 

from statistical analysis. Based on these statistical results, NOEC and LOEC values were 

determined to be 5 and 10μg/L, respectively. 

3.4 SUMMARY OF NOEC AND LOEC FOR ALL ENDPOINTS 

A summary of NOEC and LOEC for all endpoints is presented Table 20. 

4 DISCUSSION 

4.1 QUALITY OBJECTIVES AND CRITERIA 

This study achieved the minimum quality criteria specified in Appendix I, Table 3 for the 

following performance objectives: 

• Survival of A. tenuiremis in the control group was > 75% up to day 27 

• The water quality parameters (dissolved oxygen, temperature, pH, and salinity) were all 

within requirements 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

• The sex ratio in the control group was within the recommended 0.6 to 1.8 (±0.2). 

• The average number of viable offspring hatched from two successive broods in the 

control group was greater than 8 offspring/female. 

• The average number of days to the first brood was < 25 days. 

4.2 DISCUSSION OF RESULTS 

Based on a time weighted average over the 36 day exposure period, A. tenuiremis were exposed 

lindane concentrations ranging from 75 to 87% of the nominal exposure level. This range 

indicates that, on average, lindane exposure concentrations throughout the testing period were 

within 25% of the predicted level. These measured levels were calculated as time-weighted 

mean concentrations accounting for the concentration of lindane in freshly prepared test 

solutions on water change days (referred to as “UP” samples) and the aged test solutions being 

replaced (referred to as “DOWN” samples). Measured lindane concentrations in the UP samples 

ranged from 95 to 106% of the nominal levels, while test chemical in the DOWN samples ranged 

from 56 to 74% of the nominal concentrations. Possible explanations for the loss of test 

chemical are adsorption onto test chambers and sample storage bottles, as well as through natural 

degradation. 

Lindane did not affect copepod survival in the 2.5, 5, and 10μg/L test treatments throughout the 

36 day exposure period. Lindane, in contrast, significantly reduced survival of copepods exposed 

to 20μg/L by day 16 of the test, and significantly reduced copepod survival in the 15 and 20μg/L 

treatments by day 36 of the test. This was unexpected as the concentration series was specifically 

selected to allow meaningful observations of chronic developmental and reproductive endpoints 

without affecting organism survival. Copepod survival was not significantly different between 

sea water controls and the 0.05% acetone control, indicating effects observed in copepods are 

most likely attributed to the test chemical and not the solvent. The reduced survival rate might be 

attributed to increased organism stress during water changes as operators aspirated test solution 

too quickly; however, as the controls are subjected to the same stressors, this is most likely 

accounted for. 

Lindane was shown to significantly delay development time for A. tenuiremis nauplii to mature 

into the copepodite stage and subsequent transformation to the adult stage at concentrations of 

5μg/L and above. Lindane had no significant effect on sex ratio for all concentrations tested. 

Lastly, lindane did not significantly delay the time for mated females to extrude their first or 

second broods at any level tested. The most striking reproductive effect of lindane was the 

reduced number of successful mating pairs and the number of offspring that hatched. Lindane 

concentrations of 10μg/L, 15μg/L, and 20μg/L significantly reduced or completely prevented 

exposed mating pairs from successfully producing two successive viable broods. Moreover, 

lindane reduced the number of viable nauplii hatched from the first brood at the 20μg/L test level 

as well as the second brood at the 15μg/L concentration. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

5 ACKNOWLEDGEMENTS 

Our thanks to Professor G.T. Chandler of the University of South Carolina for generously 

providing BES A. tenuiremis stock mud cultures as well as growth media for algae culturing. 

Acknowledgement also needs to be made to Kate Washburn with the Chandler laboratory for 

assisting with the training of BES technical staff and for providing technical advice. Thanks to 

the staff of North Coast Laboratories LTD for analyzing the analytical chemistry data. Many 

thanks go to BES staff scientists Terry Clohesy, Nanette Bradbury, Sebastian Castro, and Amy 

Ayala for performing the studies. 

6 REFERENCES 

Un-published material:

OECD Draft Guidelines for the Testing of Chemicals. Harpacticoid Copepod Development and 

Reproduction Test with Amphiascus tenuiremis. Post June 2009. 

Published material:

ASTM E 2317-04. (2004). Standard guide for conducting renewal microplate-based life-cycle 

toxicity tests with a marine meiobenthic copepod. American Society for Testing and Materials, 

West Conshohocken, PA, 22 pp. 

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a 

control. Journal of the American Statistical Association, 50, 1,096-1,121 

Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, 20, 482-

491 

Piegorsch WW, Bailer AJ (1997) Statistics for environmental biology and toxicology. Chapman 

and Hall, London, UK 

USEPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and 

Receiving Water to Marine Organisms. EPA-800-R-02-014. 4th Edition. 

USEPA. Good Laboratory Practice Standards. 2007a. 40CFR160. 

USEPA. Good Laboratory Practice Standards. 2007b. 40CFR792. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

7 FIGURES 

 Figure 1: Layout of Microplate Test Chamber 

Test organisms are reared from nauplius to sexually mature adult in the grey shaded wells. 

Virgin male and female adults are mated in the red shaded wells. 

8 TABLES 

Table 1: Method for preparation of lindane primary stock solution 

Test chemical weight: 4.0mg

Type of solvent: Acetone

Volume of solvent used: 50mL

Test chemical primary stock concentration: 80 mg/L

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 2: Method for preparation of lindane test solutions 

Solution 

ID 

Volume of Primary Stock 

Solution used 

Volume of 

acetone used 

Percent of 

Acetone 

Carrier Used 

Volume of 

dilution 

water used 

Control 500 mL

Carrier 125μL 0.025% 500 mL

20 μg/L 125μL of primary stock solution 125 μL 0.025% 500 mL

15 μg/L 94μL of primary stock solution 94 μL 0.019% 500 mL

10 μg/L 62.5μL of primary stock solution 62.5 μL 0.013% 500 mL

5 μg/L 31.25μL of primary stock solution 31.25 μL 0.006% 250 mL

2.5 μg/L 15.6μL of primary stock solution 15.6 μL 0.003% 250 mL

Table 3: Water quality parameters: pH, salinity, dissolved oxygen 

Date Dissolved Oxygen pH Salinity 

8/16/2011 96.3 8.5 30.2 

8/19/2011 93.4 8.1 30 

8/22/2011 105.0 8.1 30 

8/25/2011 89.5 8.1 28.2 

8/28/2011 107.8 8.1 30.1 

8/31/2011 98.0 8.0 30.6 

9/3/2011 94.6 8.3 29.8 

9/6/2011 100.1 8.2 29.5 

9/9/2011 93.1 8.1 29.8 

9/12/2011 97.1 8.1 29.3 

9/15/2011 97.4 8.1 29.7 

Note: measurements were made on dilution water prior to preparation of fresh test solutions. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 4: Water quality parameter: daily temperature 

Date Temperature 

(°C) 1st read

Temperature 

(°C) 2nd read Date Temperature (°C) 1st read

Temperature 

(°C) 2nd read

8/16/2011 25.0 24.1 9/4/2011 25.0 25.0 

8/17/2011 25.0 ND 9/5/2011 25.0 25.0 

8/18/2011 ND ND 9/6/2011 25.0 25.0 

8/19/2011 25.0 24.9 9/7/2011 25.0 25.0 

8/20/2011 25.0 25.7 9/8/2011 25.0 25.0 

8/21/2011 25.0 25.0 9/9/2011 25.0 24.0 

8/22/2011 25.0 25.0 9/10/2011 25.0 25.0 

8/23/2011 25.0 25.0 9/11/2011 25.0 25.0 

8/24/2011 25.0 24.7 9/12/2011 25.0 25.0 

8/25/2011 24.5 25.0 9/13/2011 25.0 25.1 

8/26/2011 24.8 25.0 9/14/2011 25.8 25.9 

8/27/2011 24.4 24.9 9/15/2011 25.0 24.1 

8/28/2011 24.8 25.0 9/16/2011 28.0 24.0 

8/29/2011 25.0 25.0 9/17/2011 24.1 24.1 

8/30/2011 25.0 25.0 9/18/2011 27.5 26.0 

8/31/2011 25.0 25.0 9/19/2011 24.9 24.9 

9/1/2011 25.0 25.0 9/20/2011 25.0 26.0 

9/2/2011 25.0 24.9 9/21/2011 25.5 ND 

9/3/2011 25.0 25.0 

Note: Temperature was measured in dilution water from a glass bottle in environmental chamber used for study. 

ND = Not Determined 

Table 5: Summary of water quality parameters 

Temperature (°C) Dissolved 

Oxygen (%) pH Salinity (‰) 

Min 24.0 89.5 8.0 28.2 

Max 28.0 107.8 8.5 30.6 

Mean ± S.D. 25.0 ± 0.6 97.5 ± 5.3 8.16 ± 0.14 29.7 ± 0.6 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 6: Measured lindane test concentrations 

Nominal 

Concentration 

Day 0 

Up 

Day 0 

Dwn 

Day 6 

Up 

Day 6 

Dwn 

Day 12 

Up 

Day 12 

Dwn 

Day 21 

Up 

Day 21 

Dwn 

Day 27 

Up 

Day 2

Dwn 

Dilution Water 

Control <0.010 <0.010 <0.010 0.021 <0.010 0.04 <0.010 <0.010 <0.010 0.021 

Carrier Control <0.010 <0.010 <0.010 0.026 <0.010 <0.010 <0.010 <0.010 <0.010 <0.01

2.5 μg/L 2.5 1.1 2.5 1.4 2.5 1.6 2.6 1.6 2.7 1.6 

5.0μg/L 5 2.2 4.6 2.7 3.8 2.5 4.7 2.8 5.1 3.5 

10μg/L 12 5 8.6 5.6 11 6.6 9.9 5.6 7.4 14 15μg/L 15 6.9 14 7.8 17 9.8 15 ** 16 10 20μg/L 20 8.2 20 12 23 6.7 20 13 22 14 

0.5 μg/L (Spike 

1) 101% Recovery 

0.5μg/L (Spike 

2) 98.4% Recovery 

0.5μg/L (Spike 

3) 101% Recovery 

0.5μg/L (Spike 

4) 99.9% Recovery 

0.5μg/L (Spike 

5) 95.1% Recovery

Mean Spike 

recovery*** 99.1% ± 2.5% 

Note: Analysis performed by North Coast Laboratories, LTD (Arcata, California, USA) using GC-ECD.. Table cfinal report numbers 1109247 and 1109490. * Analysis could not be performed as sample was spilled during handling. ** Spike recoveries were not used to correct sample results. 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Page 27 

Table 7: Summary of measured lindane test concentration 

Nominal Concentration (μg 

lindane/L) 

Time Weighted Mean 

Measured Concentration (μg 

lindane/L) 

Percentage of Nominal (%) 

Dilution Water Control <0.01 

Carrier Control <0.01 

2.5 2.1 82.0 

5.0 3.8 75.3 

10 8.7 87.1 

15 12.6 83.9 

20 16.2 80.8 

Table 8: Survival on day 16 

Percent survival at day 16 

Treatment group Replicate Percent survival Mean Standard deviation 

plate 1 83 

Control plate 2 80 

plate 3 83 

82 2 

plate 1 83 

Carrier Control plate 2 90 

plate 3 63 

79 14 

plate 1 63 

2.5 μg/L lindane plate 2 87 

plate 3 87 

79 14 

plate 1 70 

5 μg/L lindane plate 2 50 

plate 3 87 

69 19 

plate 1 83 

10 μg/L lindane plate 2 67 

plate 3 73 

74 8 

plate 1 77 

15 μg/L lindane plate 2 63 

plate 3 57 

66 10 

plate 1 60 

20 μg/L lindane plate 2 77 

plate 3 53 

63* 12 

*Statistically significant difference in survival relative to the carrier control 
BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 9: Survival on day 36 

Percent survival at day 36 

Treatment group Replicate Percent survival Mean Standard deviation 

plate 1 77 

Control plate 2 73 

plate 3 80 

77 4 

plate 1 77 

Carrier Control plate 2 90 

plate 3 63 

77 14 

plate 1 53 

2.5 μg/L lindane plate 2 83 

plate 3 87 

74 19 

plate 1 70* 

5 μg/L lindane plate 2 43 

plate 3 83 

65 20 

plate 1 73 

10 μg/L lindane plate 2 67 

plate 3 73 

71 3.5 

plate 1 70 

15 μg/L lindane plate 2 63 

plate 3 43 

59** 14 

plate 1 60 

20 μg/L lindane plate 2 67 

plate 3 53 

60** 9 

*denotes the survival rate on day 24; the plate was accidentally dropped on day 25 during test 

maintenance. 

**Statistically significant difference in survival relative to the carrier control 

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Table 10: Time to transformation to copepodite stage 

Number of days to transformation to copepodite stage 

Treatment group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 9.6 

Control plate 2 8.9 

plate 3 9.9 

9.5 0.5 

plate 1 9.2 

Carrier Control plate 2 9.0 

plate 3 9.6 

9.3 0.3 

plate 1 10.0 

2.5 μg/L lindane plate 2 10.0 

plate 3 10.0 

10.0 0.0 

plate 1 12.5 

5 μg/L lindane plate 2 11.5 

plate 3 11.1 

11.7* 0.7 

plate 1 14.3 

10 μg/L lindane plate 2 13.3 

plate 3 14.1 

13.9* 0.5 

plate 1 17.0 

15 μg/L lindane plate 2 14.3 

plate 3 15.4 

15.6* 1.4 

plate 1 14.4 

20 μg/L lindane plate 2 16.1 

plate 3 14.5 

15.0* 1.0 

*Statistically significant difference when compared to the Carrier Control 

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Table 11: Time in copepodite stage 

Number of days in copepodite stage 

Treatment group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 7.9 

Control plate 2 8.3 

plate 3 8.1 

8.1 0.2 

plate 1 8.9 

Carrier Control plate 2 7.9 

plate 3 7.9 

8.2 0.6 

plate 1 8.7 

2.5 μg/L lindane plate 2 8.3 

plate 3 8.6 

8.5 0.2 

plate 1 8.5 

5 μg/L lindane plate 2 7.9 

plate 3 7.9 

8.1 0.3 

plate 1 8.5 

10 μg/L lindane plate 2 9.2 

plate 3 8.6 

8.8 0.4 

plate 1 10.0 

15 μg/L lindane plate 2 8.4 

plate 3 9.9 

9.4 0.9 

plate 1 9.4 

20 μg/L lindane plate 2 8.9 

plate 3 8.3 

8.9 0.6 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

Table 12: Time to transformation to adult stage 

Number of days to transformation to adult stage 

Treatment Group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 16.9 

Control plate 2 17.3 

plate 3 18.0 

17.4 0.6 

plate 1 18.3 

Carrier Control plate 2 17.0 

plate 3 17.2 

17.5 0.7 

plate 1 18.1 

2.5 μg/L lindane plate 2 18.1 

plate 3 18.6 

18.3 0.3 

plate 1 20.7 

5 μg/L lindane plate 2 20.0 

plate 3 19.0 

19.9* 0.9 

plate 1 23.2 

10 μg/L lindane plate 2 22.5 

plate 3 22.4 

22.7* 0.4 

plate 1 26.4 

15 μg/L lindane plate 2 22.9 

plate 3 24.6 

24.6* 1.8 

plate 1 23.8 

20 μg/L lindane plate 2 24.5 

plate 3 22.8 

23.7* 0.9 

*Statistically significant when compared to the Carrier Control 

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Table 13: Sex ratio 

Sex ratio 

Treatment group Replicate Number 

of males 

Number 

of 

females 

Ratio 

(female:male) 

Total 

Mean 

Standard 

deviation 

plate 1 35 65 1.88 

plate 2 44 56 1.25 

plate 3 48 52 1.09 Control 

Plate 1, 2, 

and 3 27 37 

1.4 0.4 

plate 1 53 47 0.90 

plate 2 36 64 1.75 

Carrier Control plate 3 53 47 0.89 

Plate 1, 2, 

and 3 27 21 

1.2 0.5 

plate 1 79 21 0.27 

plate 2 64 36 0.57 

plate 3 33 67 2.00 2.5 μg/L lindane Plate 1, 2, 

and 3 31 23 

0.9 0.9 

plate 1 80 20 0.25 

plate 2 64 36 0.57 

plate 3 32 68 2.13 5 μg/L lindane Plate 1, 2, 

and 3 28 24 

1.0 1.0 

plate 1 35 65 1.86 

plate 2 62 38 0.63 

plate 3 47 53 1.11 10 μg/L lindane 

Plate 1, 2, 

and 3 25 28 

1.2 0.6 

plate 1 45 55 1.22 

plate 2 31 69 2.25 

plate 3 50 50 1.00 15 μg/L lindane 

Plate 1, 2, 

and 3 20 26 

1.5 0.7 

plate 1 67 33 0.50 

plate 2 63 37 0.58 

20 μg/L lindane plate 3 45 55 1.20 

Plate 1, 2, 

and 3 28 18 

0.8 0.4 

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Table 14: Time to first egg extrusion since date of pairing 

Time to first brood (days) since date of pairing 

Treatment group Replicate Mean number of 

days 

Total 

Mean Standard deviation 

plate 1 2.0 

plate 2 3.0 Control 

plate 3 2.2 

2.4 0.5 

plate 1 4.0 

Carrier Control plate 2 1.0 

plate 3 1.8 

2.3 1.5 

plate 1 3.7 

2.5 μg/L lindane plate 2 2.3 

plate 3 1.0 

2.3 1.4 

plate 1 ** 

5 μg/L lindane plate 2 4.6 

plate 3 2.8 

3.7 1.3 

plate 1 2.3 

10 μg/L lindane plate 2 1.0 

plate 3 1.0 

1.4 0.8 

plate 1 2.7 

15 μg/L lindane plate 2 NC 

plate 3 4.0 

3.3 0.9 

plate 1 2.0 

20 μg/L lindane plate 2 NC 

plate 3 4.0 

3.0 1.4 

** Plate dropped by technician the day after mating 

NC=Could not be calculated since no females became gravid 

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Table 15: Number of nauplii hatched from first brood 

Number of nauplii hatched from first brood 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 6.3 

plate 2 4.9 Control 

plate 3 6.4 

5.9 0.8 

plate 1 6.7 

Carrier Control plate 2 4.5 

plate 3 6.3 

5.8 1.2 

plate 1 4.3 

2.5 μg/L lindane plate 2 6.3 

plate 3 4.0 

4.9 1.3 

plate 1 ** 

5 μg/L lindane plate 2 3.0 

plate 3 4.8 

3.9 1.3 

plate 1 3.5 

10 μg/L lindane plate 2 1.0 

plate 3 4.0 

2.8 1.6 

plate 1 7.0 

15 μg/L lindane plate 2 0.0 

plate 3 0.0 

2.3 4.1 

plate 1 1.0 

20 μg/L lindane plate 2 0.0 

plate 3 0.0 

0.3* 0.6 

*Statistically significant difference when compared to the Carrier Control 

** Plate dropped by technician the day after mating 

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Table 16: Time to second brood since date of pairing 

Time to second brood (days) since date of pairing 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 2.5 

plate 2 1.8 Control 

plate 3 2.2 

2.2 0.4 

plate 1 2.3 

Carrier Control plate 2 2.0 

plate 3 2.5 

2.3 0.3 

plate 1 1.5 

2.5 μg/L lindane plate 2 2.0 

plate 3 5.0 

2.8 1.9 

plate 1 *** 

5 μg/L lindane plate 2 2.2 

plate 3 1.0 

1.6 0.8 

plate 1 3.0 

10 μg/L lindane plate 2 3.0 

plate 3 2.0 

2.7 0.6 

plate 1 3.5 

15 μg/L lindane plate 2 NC 

plate 3 NC 

3.5 0 

plate 1 NC 

20 μg/L lindane plate 2 NC 

plate 3 NC 

NC NC 

*** Plate dropped by technician the day after mating 

NC=Could not be calculated since no females became gravid 

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Table 17: Number of nauplii hatched from second brood 

Number of nauplii hatched from second brood 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 5.1 

plate 2 5.5 Control 

plate 3 7.3 

6.0 0.8 

plate 1 7.3 

Carrier Control plate 2 7.0 

plate 3 5.8 

6.7 1.2 

plate 1 4.5 

2.5 μg/L lindane plate 2 5.7 

plate 3 ** 

5.1 1.3 

plate 1 *** 

5 μg/L lindane plate 2 5.3 

plate 3 7.4 

6.4 1.3 

plate 1 4.0 

10 μg/L lindane plate 2 5.0 

plate 3 10.0 

6.3 1.6 

plate 1 0.0 

15 μg/L lindane plate 2 0.0 

plate 3 0.0 

0* 0 

plate 1 0.0 

20 μg/L lindane plate 2 0.0 

plate 3 0.0 

0* 0 

*Statistically significant difference when compared to the Carrier Control 

** Lone female on plate extruded second brood sac but did not hatch eggs prior to accidentally 

being removed from the test well by a technician during a water change. 

*** Plate dropped by technician the day after mating 

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Table 18: Total number of nauplii that hatched from two successive broods 

Total number of nauplii hatched from two successive broods 

Treatment group Replicate Mean number of 

nauplii 

Total 

Mean Standard deviation 

plate 1 11.4

Control plate 2 9.6

plate 3 14.1

11.7 2.3 

plate 1 14.0

Carrier Control plate 2 11.5

plate 3 12.0

12.5 1.3 

plate 1 9.0

2.5 μg/L lindane plate 2 11.7

plate 3 0**

6.9 6.1 

plate 1 ***

5 μg/L lindane plate 2 8.3

plate 3 12.2

10.3 2.8 

plate 1 8.0

10 μg/L lindane plate 2 6.0

plate 3 14.0

9.3 4.2 

plate 1 7.0

15 μg/L lindane plate 2 0**

plate 3 0**

4.1 2.3 

plate 1 0**

20 μg/L lindane plate 2 0**

plate 3 0**

0 0 

* Statistically different when compared to the Carrier Control 

** Replicates with no females having two broods 

*** Plate dropped by technician the day after mating 

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Table 19: Fertilization success 

Fertilization success: mating pairs able to produce two successive broods that hatched 

Treatment Replicate 

Number of 

successful 

pair 

Total number 

of pairs 

% successful 

mating pair Mean Standard deviation 

Control PLATE 1 11 11 100% 

Control PLATE 2 6 8 75% 

Control PLATE 3 8 10 80% 

85% 13% 

Carrier control PLATE 1 3 5 60% 

Carrier control PLATE 2 2 4 50% 

Carrier control PLATE 3 4 6 67% 

59% 8% 

2.5 μg/L lindane PLATE 1 2 2 100% 

2.5 μg/L lindane PLATE 2 3 6 50% 

2.5 μg/L lindane PLATE 3   **  

75% 35% 

5μg/L lindane PLATE 1 *** *** *** 

5μg/L lindane PLATE 2 4 7 57% 

5μg/L lindane PLATE 3 5 7 71% 

64% 10% 

10μg /L lindane PLATE 1 1 9 11% 

10μg /L lindane PLATE 2 1 8 13% 

10 μg /L lindane PLATE 3 1 10 10% 

11%* 1% 

15μg/L lindane PLATE 1 0 10 0% 

15μg/L lindane PLATE 2 0 7 0% 

15μg/L lindane PLATE 3 0 4 0% 

0%* 0% 

20 μg/L lindane PLATE 1 0 7 0% 

20μg/L lindane PLATE 2 0 7 0% 

20μg/L lindane PLATE 3 0 7 0% 

0%* 0% 

* Denotes statistically significant difference when compared to the solvent control 

** Lone female on plate extruded second brood sac but did not hatch eggs prior to accidentally 

being removed from the test well by a technician during a water change. 

*** Plate dropped by technician the day after mating 

Note: The total number of pairs listed in this table is derived from mating pairs that could be 

observed until the end of the test or until they produced two broods that hatched, whichever 

came first. A single mating pair in both control replicate #2 and 2.5μg/L replicate #2 was 

excluded because the second brood had extruded but did not hatch prior to test termination on 

Day 36. 

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Table 20: Summary table of NOEC and LOEC for all endpoints 

Endpoint NOEC in μg lindane /L LOEC in μg lindane /L

Day 16 survival (%) 15 20 

Day 27 survival (%) 10 15 

Time to copepodite stage 2.5 5 

Time in copepodite stage >20 >20 

Time to adult 2.5 5 

Sex ratio >20 >20 

Time to 1st Brood 20 >20 

Time to 2nd Brood >15 >15 

Number offspring 1st Brood 15 20 

Number offspring 2nd Brood 10 15 

Number of mating pair with successful 

reproduction 5 10 

Number nauplii hatched over two clutches 10 15 

Note: 2.5 μg lindane /L was the lowest concentration tested and 20 μg lindane /L was the highest 

concentration tested. 

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10 APPENDIX II: QUALITY OF LABORATORY WATER AT BES 

Baseline chemical analyses of laboratory reagent water used for preparation of 

synthetic seawater and lindane test solutions at the BES laboratory in 2011 

Samples collected 05/10/2011 by BES staff and analyzed on 5/12/2011 by Basic Laboratory 

(Redding, California, USA) 

Chemical/Constituent 

Laboratory 

Processed 

Reagent 

Water 

Chemical/Constituent 

Laboratory 

Processed 

Reagent 

Water 

Cobalt (μg/L) <0.1 Chlordane (μg/L) <0.05 

Copper (μg/L) 2.30 Dieldrin (μg/L) <0.010 

Iron (μg/L) <100 Endosulfan I(μg/L) <0.010 

Zinc (μg/L) 5.0 Endosulfan II(μg/L) <0.020 

Aluminum (μg/L) 5.3 Endosulfan Sulfate(μg/L) <0.020 

Arsenic (μg/L) <0.5 Endrin(μg/L) <0.010 

Cadmium (μg/L) 0.05 Endrin aldehyde(μg/L) <0.010 

Chromium (μg/L) 0.2 Heptachlor(μg/L) <0.010 

Lead (μg/L) 0.3 Heptachlor epoxide(μg/L) <0.010 

Mercury (μg/L) <0.2 Methoxychlor(μg/L) <0.010 

Nickel (μg/L) <1.0 4,4-DDT(μg/L) <0.010 

Silver (μg/L) <0.5 4,4-DDE(μg/L) <0.010 

TOC (mg/L) 0.2 4,4-DDD(μg/L) <0.020 

alpha-BHC(μg/L) <0.010 2,4-DDT(μg/L) <0.010 

beta-BHC(μg/L) <0.005 2,4-DDE(μg/L) <0.010 

gamma-BHC(μg/L) <0.010 2,4-DDD(μg/L) <0.020 

delta-BHC(μg/L) <0.005 Toxaphene(μg/L) <0.50 

Aldrin (μg/L) <0.005 

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11 APPENDIX III: LINDANE CHEMISTRY DATA REPORT 

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12 APPENDIX IV: STUDY PROTOCOL (P27569) 

PROTOCOL 

Effect of Lindane on the Survival, Development, and Reproduction 

of the Marine Copepod Amphiascus Tenuiremis

 Prepared for: RTI International 

 Protocol Number: P27569 

 BES Study Number: 27569 

 PROTOCOL WRITTEN BY: NANETTE BRADBURY, B.SC. 

 MANAGEMENT APPROVAL: DAVID S. BLOCK, PH.D. 

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PROTOCOL APPROVAL (STUDY NO: 27569) 

Pages 1 - 13 

____________________________________ ___April 21, 2011___ 

David S. Block Date 

Study Director 

Block Environmental Services, Inc. 

____________________________________ ____________________ 

Carol Sloan Date 

Sponsors Agent 

RTI International 

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SCHEDULE OF WORK 

STUDY TITLE Effect of Lindane on the Survival, Development, and 

Reproduction of the Marine Copepod Amphiascus Tenuiremis

RELEVANT 

GUIDELINE 

Draft OECD Test Guideline ‘Harpacticoid copepod development 

and reproduction test with Amphiascus tenuiremis’

STUDY NUMBER 27569 

SPONSOR 

(name & address) 

Hernan Navarro, Ph.d.

RTI International

Hermann 120

3040 Cornwallis Rd

RTP, NC 27709, USA.

SPONSORS AGENT 

(name & address) 

Carol Sloan

RTI International

Hermann 120

3040 Cornwallis Rd

RTP, NC 27709, USA.

STUDY DIRECTOR David S. Block, Ph.D

REPORT DUE TO 

SPONSOR 15 December 2011

TEST SUBSTANCE 

& CAS NUMBER 

Lindane CAS# 58-89-9

PROPOSED 

EXPERIMENTAL 

DATES 

START: April 18, 2011 COMPLETION: May 6, 2011

[Amended]

START: August 16, 2011 COMPLETION: September 21, 2011

PRINCIPLE 

SCIENTIST(S) 

Terence Clohesy

Nanette Bradbury

ASSISTANT 

SCIENTIST(S) 

Sebastian Castro

Amy Ayala

MAIN TEST 

LOCATION 

2451 Estand Way, Pleasant Hill, California, USA

Testing Room #4

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SCHEDULE OF WORK 

STANDARD 

OPERATING 

PROCEDURES 

All SOPS used will be documented in the study book. 

ANALYTICAL 

CHEMISTRY METHOD 

Lindane will be measured in test solutions using USEPA Method 505 

TEST 

CONCENTRATION(S) 

TO BE EMPLOYED 

The test concentrations will be a dilution water control, solvent control 

and 2.5, 5, 10, 15 and 20 μg/L. 

CIRCULATION LIST Sponsor or Sponsor’s Agent 

Client Manager 

QA 

Principal Scientists 

Assistant Scientists 

INTRODUCTION 

The objectives of this study are to determine the effect of lindane on the survival rate, 

development, mating success, and reproductive output of the copepod Amphiascus tenuiremis. 

This method is based on a draft OECD Test Guideline ‘Harpacticoid copepod development and 

reproduction test with Amphiascus tenuiremis’ (OECD, 2009) and the published ASTM 

Guideline entitled “Standard guide for conducting renewal microplate-based life-cycle 

toxicity tests with a marine meiobenthic copepod.” (ASTM, 2004). 

Principles of the test

Newly hatched nauplii (< 24 hours old) will be exposed in individual microwell (250 μL total 

volume) test chambers to a range of lindane concentrations in seawater at 25°C for a period of 36 

days. Once maturing into sexually dimorphic virgin adults (typically between 15-18 days at 

25 ̊C), male and female copepods will be paired within treatment in order to determine the effect 

of lindane on mating success and offspring production. The test will be terminated when all 

mating pairs have successfully produced two viable broods or on Day 36 of the exposure period, 

whichever comes first. All phases of the test will be performed under semi-static test conditions 

with water renewal occurring every three days. The test duration should allow sufficient time for 

the control animals to reach adulthood, mate and produce at least two clutches of offspring. 

Test endpoints, such as the survival rate, time to developmental milestone, mating success, and 

offspring production will be statistically compared between lindane exposed copepods and 

laboratory controls in order to calculate no observed effect (NOEC) and lowest observed effect 

(LOEC) concentrations. 

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MATERIALS 

Test Chemical

A lindane certificate of analysis and purity information will be supplied by the Sponsor. 

The Sponsor will outline all precautions required in the handling, storage and disposal of lindane. 

Unused lindane will be resubmitted back to the Sponsor upon completion of the study. 

TEST ORGANISM 

The test organism will be the marine crustacean Amphiascus tenuiremis. The copepods will 

originate from laboratory cultures established by Dr Tom Chandler, Professor of Environmental 

Health and Marine Sciences, University of South Carolina, Columbia, SC 29208 who will 

confirm the taxonomic identity of this species. 

Test animals should be derived from a healthy stock (i.e., showing no signs of stress such as high 

mortality, poor fecundity etc). The stock animals must be maintained in culture conditions 

(light, temperature, seawater medium and feeding regimen) similar to those to be used in the test. 

Justification for the selection of the test species

Amphiascus tenuiremis’c.f. Mielke is an easily-cultured, diosaccid harpacticoid copepod that is 

amphi-Atlantic in distribution ranging from the North Sea/Baltic intertidal to the southern Gulf 

of Mexico (Ref 2). It is an epifaunal to deep burrowing (0 to 15 mm) species that ingests 

sediments for diatoms and bacteria, and produces burrowing larvae with no swimming ability. 

Sexes are dimorphic with females reaching 0.4 mm in length and males 0.25 to 0.30 mm. Males 

are also streamlined in shape and have a swollen geniculate segment on their first antenna to 

clasp the female. 

Amphiascus tenuiremis’ has a generation time (egg to egg) of 17 - 18 days at 25°C in microplate 

culture, or 14 - 15 days in 25°C sediment culture. At 21°C in sediments, the generation time is 

20 - 21 days egg to egg. Females are sexually mature after the fifth copepodite stage (that is, in 

the twelfth life stage). They then mate and produce their first clutch in two to three days. Sperm 

transfer is via a membranous spermatophore sac attached externally by the male to the female 

genital pore. Isolated virgin females can be fertilized successfully up to 14 days post adulthood, 

and females will not extrude viable eggs unless paired with a reproductively-functional mature 

male. Females will produce eight to nine clutches over their median lifetime of 49 ± 2 days. 

Nauplii hatch within two to three days of egg extrusion and reach the copepodite stage in six to 

seven days. 

CULTURE MEDIUM 

Natural or synthetic seawater may be used as the test medium. If natural seawater is used, it 

should be collected from a location distant from any known sources of pollution and filtered to 

remove indigenous organisms. If synthetic seawater is used, it should be prepared by dissolving 

reagents of recognized analytical grade, or a commercially available formulation, in distilled or 

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deionized water. Any water in which the copepods show suitable long-term survival with 

normal development and fecundity may be used as the test medium. 

The salinity of the dilution water will be 30 ± 3‰. The dilution water must have a dissolved 

oxygen concentration above 70% of the air saturation value, and a pH of 8.0 ± 0.4 before being 

used to prepare the test solutions. The dilution water should be aerated for 24 hours before use. 

The dilution water will be filtered to 0.2 μm prior to test solution preparation to minimize 

bacterial contamination. 

The dilution water will be of constant quality and measurement of heavy metals (e.g., Cu, Pb, 

Zn, Hg, Cd, Ni), major anions and cations (e.g., Ca, Mg, Na, K, Cl, SO4), pesticides (e.g., total 

organophosphorus and total organochlorine pesticides), total organic carbon and suspended 

solids should be made. 

TEST APPARATUS 

Disposable polystyrene microwell plates will be used as test vessels. Polystyrene plates are not 

ideal for testing organic compounds because of non-specific binding which may reduce the 

chemical availability of the test compound. The polystyrene microplates will therefore be precoated with glass or polyacrylamide gel unless it can be demonstrated that there is no significant 

loss of the test compound between water renewals using ‘uncoated’ polystyrene microwell 

plates. The microplates will be 96 well plates each well having a 300 μL total volume and a 250 

μL working volume. 

EXPERIMENTAL DESIGN 

The test solutions will be maintained at 25 ± 2°C by housing the test vessels in a temperaturecontrolled room or in climate controlled chambers. Glass or plastic ‘humidity chambers’ will be 

used to reduce evaporative losses. Potential for evaporative loss will be measured before 

beginning any tests by placing seawater filled microplates in the test room or chamber for 72 

hours and measuring salinity change over this period. Photoperiod will be controlled to provide 

12 h light: 12 h dark. 

In addition, some or all of the following equipment will be required: 

• oxygen meter (with microelectrode or other suitable equipment for measuring 

dissolved oxygen in low volume (e.g., 10-mL) samples); 

• pH meter; 

• conductivity meter, osmometer or salinity refractometer; 

• adequate apparatus for the control of the lighting regime; 

• stereo compound microscope, preferably inverted with dark-field or Hoffman DIC 

illumination; 

• dissection stereomicroscope, preferably with dark-field illumination and fiber 

optic episcopic illumination. 

• climate-controlled environmental chambers or a temperature regulated room; 

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• 0.5 m3 glass or plastic rectangular containers with sealable lids to create humidity 

chambers for the microplates (to minimize evaporative loss). 

• various plastic or stainless-steel mesh sieves (0.05 mm to 0.25 mm) to culture, 

capture and sort copepods; 

• Pasteur pipettes, preferably silanized or equivalent treatment to prevent copepods 

from adhering to pipette walls; 

• adjustable single and multi-channel volumetric pipettors (1-10 μL, 100-500 μL) 

for food additions; 

• 250 μL Hamilton glass volumetric syringes with 23 gauge beveled stainless-steel 

needles; 

• haemacytometer cell counting chamber or electronic particle counter/analyser 

(e.g., a Coulter principle impedance-based system) 

Development to adult

Nauplii (< 24 hours old) will be obtained by isolating ovigerous females from a mass culture at 

least 3 weeks old. Nauplii will be exposed in individual microwells of a 96 microwell plate (30 

animals per microplate) with three microplates per treatment (total of 90 animals) for the control, 

solvent control and each test concentration in the definitive test. The outer microwells of the 

plate will be left empty. 

The positions of the microplates in the test area will be randomized. Addition of nauplii, 

subsequent handling, loading, observation and feeding will be performed in an irregular plate 

order. If experimental units are handled in treatment or concentration order, then some timerelated effect, such as operator fatigue or other error, could lead to greater or lesser effects at the 

higher or lower concentrations. 

The number of days it takes for development of each individually isolated copepod, from a first 

stage nauplius to a juvenile copepodite to a reproductively mature adult, will be checked daily. 

Survival of larvae (nauplii), juveniles (copepodites) and all adults will also be recorded daily up 

to and including the last day of the test. The sex of each adult will be recorded after sexual 

dimorphism is reached. 

Creation of sexed pairs

On the first test solution renewal day upon reaching adulthood, when the individual copepods 

can be sexed visually (after 15 - 18 days), mature virgin males and females will be removed from 

their respective treatment or control microplates and paired impartially with an opposite sex 

individual (i.e., from the same microplate within the same treatment) in a new or unused 

microwell filled with test medium for each treatment or control replicate. If necessary the males 

and females may be transferred to a separate vessel containing the appropriate test solution (or 

control) media to aid identification of sex. If not all the animals have achieved sexual maturity at 

this renewal day then unpaired animals will be left until the next renewal day and paired with 

copepods that have become adult during the preceding three days. 

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If survival was 100% and the sex ratios are balanced then this would result in 15 pairs. If sex 

ratios are skewed, or mortality rates have produced insufficient numbers of males and/or females 

to produce multiple mating pairs within a given microplate within a given treatment, then virgin 

adults may be mated across microplates within treatments. However, this must be noted in any 

report of results, and an a priori assumption of no significant microplate to microplate variability 

within the given treatment(s) must be made by the investigator. 

Once individual mating pairs are established for each treatment and control adults, 90-95% of 

transfer seawater will be aspirated and replaced with the appropriate control or test substance 

concentration. This replacement of media after matings are in place will help to ensure that 

transfer seawater is diluted to negligible levels at the beginning of the mating time window. In 

the case of uneven sex ratios, spare unmated males or females may be retained and monitored for 

survival as individuals to the end of the test exposure period. 

Offspring production

After pairing of male: female mating pairs, observations will be made daily for the presence of 

females carrying their first and subsequent egg sacs. In controls, two fertile egg sacs per female 

(> 4 eggs/sac normally) are usually extruded simultaneously from the abdomen in 1 - 2 days. 

Total egg number per mating pair will be counted by direct observation of all eggs in each egg 

sac if desired. Numbers of successfully hatching nauplii (i.e., total viable clutch, or fecundity) 

will be recorded for the first and second successive clutches of eggs. Hatching number can be 

obtained by subtracting any non-hatched or necrotic eggs from the total clutch size at the clutchone and clutch-two time points. Alternatively, numbers of hatched nauplii crawling on the 

bottom of the microwell may be counted by direct microscopic observation. 

Once the second clutch is hatched, then they will be counted and the bioassay is complete for a 

given mating pair. All such mating pairs should be recorded as a “successful mating”. Percent 

mating success will be calculated for each group of mating pairs in each microplate within each 

treatment and control as the ratio of successful mating pairs versus unsuccessful pairs (i.e., those 

pairs unable to produce 2 viable clutches of eggs). An average percent mating success and 

standard deviation will be calculated from these data for each treatment and control. The mean 

number of viable offspring produced through two broods should be > 8 offspring per mating pair 

in the controls. At the end of the exposure period, all remaining copepods (i.e., including 

unfertilized females and males) will be discarded. 

Observations

Copepod lifecycle endpoints will be monitored daily in each microplate. Test observations 

include mean development rates from nauplius to the copepodite and adult stages, respectively, 

fertilization success (i.e., number of mating pairs able to produce two successive clutches), total 

mean viable offspring production per mating pair (i.e. through two clutches averaged over the 

number of mating pairs created and tested), time to production of first clutch, time interval 

between successive clutches, and stage-specific mortalities. Observations may also be made for 

any abnormal behaviour (relative to the controls), including uncoordinated swimming, loss of 

equilibrium, change in pigmentation and atypical quiescence. Sex ratio may be determined by 

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observations of male and female secondary sexual characteristics which in harpacticoid 

copepods normally can be distinguished in the 5th copepodite stage at the earliest. Numbers of 

aborted egg sacs and necrotic/infertile eggs may also be counted. 

Feeding

The test animals will be fed on Day 0 and at every other microwell renewal day (every 6th day of 

the test) with cultured algae. Each microwell will receive the equivalent of 2 μl of a 1:1:1 

volume mixed-algal cell suspension (centrifuged, pelletized and re-constituted in fresh seawater 

to 107 cells/mL) of a chlorophyte (e.g., Dunaliella tertiolecta), a chrysophyte (e.g., Isochrysis 

galbana) and the diatom Phaeodactylum tricornutum. Therefore the cell density added to each 

test microwell at each feeding should target ~ 2 x 104

 cells/microwell. This density will support 

copepod maturation and reproduction without fouling the microwell environment. 

Test medium renewal

The test will be run as a semi-static renewal with the medium in each test microwell being 

renewed every third day of the test. Potential evaporation losses of media from the test chambers 

will be monitored daily and, should not exceed 10% on a daily basis. To prevent such a 

scenario, microplates will be placed over a platform over a water bath inside a humidity chamber 

in the temperature regulated incubator. If necessary excess evaporative losses can be corrected 

by more frequent medium renewal. 

When the medium is renewed, a fixed volume (preferably 90-95% of the total test medium 

volume) will be aspirated from each microwell chamber under dissection stereomicroscopic 

observation and cool fiber optic or tangential lighting using a Hamilton gas syringe of suitable 

needle diameter (e.g., 23 gauge bevelled). Care should be taken that copepods are not aspirated 

into the syringe. If copepods are aspirated they may experience stress or egg sac damage; thus 

such aspirated copepods should be disqualified from the test. 

PREPARATION OF TEST SOLUTIONS 

Stock solutions of lindane will be prepared in an organic solvent (acetone). The test solutions 

will be prepared by addition of a stock solution to dilution water and each test concentration and 

the solvent control will contain the same concentration of solvent, which will not exceed 1 ml/L. 

The volume of each test solution prepared will be in excess of that required for the replicate test 

vessels to allow for initial physical and chemical analysis. Further details of the test solution 

preparation will be documented in the study book. 

PHYSICAL AND CHEMICAL ANALYSIS 

During the test, dissolved oxygen, pH and temperature should be measured in the controls and all 

test concentrations each time test medium is renewed. As a minimum, these measurements 

should be made in the control(s) and in the highest test concentration. Temperature should 

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preferably be monitored continuously in at least one blank test microplate or in a 100-mL Griffin 

beaker filled with deionized water and placed inside the microplate humidity chamber.

The analytical chemistry method used will be USEPA Method 505 “Analysis of Organohalide 

pesticides and Aroclors in Drinking Water”. Chemical analysis will be conducted on all newly 

prepared solutions of controls and all test concentrations (Ups) and on the solutions removed 

from the microwells at test renewal (Downs). Chemical analysis will be conducted at the start of 

the test and then on at least one water renewal per week for the duration of the test. If timings 

between test renewals varies then a mixture of different length renewals will be included in the 

analysis (e.g. 2 or 3 days between renewals). 

BIOLOGICAL DATA ANALYSIS 

The mortality data for selected time periods will be analyzed to calculate the median effect 

concentrations (EC50) and 95% confidence intervals. 

Fisher’s Exact Test or Pearson’ Chi-square goodness of fit tests (Ref) can be used to identify 

significant differences in survival between the control(s) and each treatment after selected time 

periods (e.g. 10 and 21 days). 

Larval development rates, stage specific mortality, percent mating success and sex ratios may all 

be analyzed for significant differences relative to the controls. Reproduction data, expressed as 

the mean number of F1 offspring produced from fertilized females (F0) in each replicate will be 

calculated. Generally, the reproduction data are not adjusted for mortality but are presented on a 

per female or per mating pair basis. If an animal dies after producing young, the total number of 

offspring produced up to the time of death will be used. On occasion, it may be necessary to 

make an adjustment for mortality and those animals that died during the exposure will not be 

included in the statistical analysis. One-way nested analysis of variance (ANOVA) will be used 

to evaluate effects of the test substance on the reproduction (and other chronic endpoints) of the 

copepods, and a multiple comparison procedure (e.g., Dunnett's test) will be used to define the 

treatment level resulting in e.g., significantly different reproduction from the control(s) (p = 

0.05). For ANOVA, individual microwell mating pairs will be nested within their particular 

microplate, which will be replicated within a given treatment. A nested model will be ideal if 

data are balanced across microplates and treatments because microwells within a microplate are 

not strictly independent of one another. 

Analysis of variance is a parametric procedure and is based on the assumptions that the 

observations are independent and normally distributed, and that the variance of the observations 

is homogeneous across all test substance treatments and the control(s). Data will be tested for 

normality and homogeneity of variance prior to analysis of variance techniques. Data that fail to 

meet these assumptions for ANOVA will be analyzed using non-parametric (distribution-free) 

procedures. 

The biological data may also be analyzed using a regression model in order to estimate the 

concentration that would cause an x% reduction in reproductive output, i.e., ECx (e.g., EC50, 

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EC20 or EC10). Depressed intrinsic or instantaneous rates of population increase (rm or λ), may 

also be calculated. Details of these calculations will be recorded in the study book. 

If a statistically significant difference in survival, development rate, or reproduction is detected 

between the control and solvent control, only the solvent control will be used as the basis for the 

calculation of results. If no significant differences exist between control and solvent control data, 

these may be pooled for greater statistical power in comparisons of control response with test 

substance treatments. 

For the test to be valid, the following performance criteria will be met: 

Parameter Units Expected Results

Survival: Daily assessment of survival will 

be made to provide a basis for expression 

and interpretation of reproductive output. 

Not 

Applicable 

75% or greater survival in controls. No individual control 

microplates survival should be lower than 70%. At the end of 

the exposure period. 

Dissolved Oxygen Concentration: 

Expressed as % of the air saturation value. Percentage 

The dissolved oxygen concentration must have been at least 

70% of the air saturation value (ASV) throughout the exposure 

period. 

Temperature: Low variation in exposure 

temperature. needed o C The temperature should not vary more than 4 o C during the entire test with a 36 day mean temperature of 25 ± 2 o C. 

pH: Low variation in exposure pH needed. pH units pH must not vary more than 0.5 units from the average control 

pH with a 36 day mean pH of 8.0 ± 0.4. 

Conductivity/salinity: Low variation in 

conductivity/salinity needed. % Conductivity/salinity must not vary more than 10% from the control start value of 30‰ 

Sex Ratio: Number The sex ratio in F0 controls (females/males) should on the 

average range between 0.4 to 2.0. 

Viable Offspring: Number The average number of viable offspring produced through two 

clutches in the control(s) should not be less than 8 individuals. 

Average Number of Days to the First 

Clutch Number 

The average number of days to the first clutch in the control(s) 

should at a room temperature of 25oC not exceed the number of 

days representing 70% of the total exposure period or 25 days, 

whichever is less. 

REPORTING 

Following completion of the experimental phase and internal scientific review, a draft report will 

be submitted to the Sponsor for comment. The content and format of the draft report will 

comply with the requirements of the relevant regulatory authorities. Any amendments to the 

content and format of the draft report requested by the Sponsor that are in addition to the 

requirements of relevant regulatory authorities will incur an extra charge. Unless the Sponsor 

requests any amendments within three months of receipt of the draft report, the project shall be 

deemed completed and final reports issued. 

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BES Project No. 27569 EPA Contract No. EP-W-06-026 

COMPLIANCE 

GLP compliance

The study will be conducted in accordance with USEPA Good Laboratory Practice Regulations. 

Quality assurance evaluation

The study described in this protocol will be subject to Quality Assurance Audit. The form of this 

inspection will be described in the QA Standard Operating Procedures.

ARCHIVING PROCEDURES 

 

All raw data and other relevant documents from this study will be archived, identified by the 

study number. 

REFERENCES 

ASTM E 2317-04. (2004). Standard guide for conducting renewal microplate-based life-cycle 

toxicity tests with a marine meiobenthic copepod. American Society for Testing and Materials, 

West Conshohocken, PA, 22 pp. 

OECD Draft Guidelines for the Testing of Chemicals. Harpacticoid Copepod Development and 

Reproduction Test with Amphiascus tenuiremis. Post June 2009. 

Lang, K (1948) Monographie der Hapacticiden. Hanken Ohlsson, Lund, Sweden 

Dunnett, C.W. (1955). A multiple comparison procedure for comparing several treatments with a 

control. Journal of the American Statistical Association, 50, 1,096-1,121 

Dunnett, C.W. (1964). New tables for multiple comparisons with a control. Biometrics, 20, 482-

491 

Piegorsch WW, Bailer AJ (1997) Statistics for environmental biology and toxicology. Chapman 

and Hall, London, UK 

USEPA. 2002. Short-term Methods for Estimating the Chronic Toxicity of Effluents and 

Receiving Water to Marine Organisms. EPA-800-R-02-014. 4th Edition. 

USEPA. Good Laboratory Practice Standards. 2007a. 40CFR160. 

USEPA. Good Laboratory Practice Standards. 2007b. 40CFR792. 

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Page 127 

13 APPENDIX V: DEVIATIONS FROM STUDY PROTOCOL 

13.1 LIST OF DEVIATIONS

Below is a list of the deviations encountered during the study.

Deviation

number

Deviation title Deviation description

1 Protocol not distributed or

signed.

The completed test protocol was not submitted to Sponsor or

signed by Sponsor prior to initiation of study.

2 Starting number of 

nauplii/Empty test wells 

On D1, several wells across all test treatments were observed

to contain >1 nauplius per well.  These extra nauplii were

transferred to test wells observed to be empty on the same

replicate within each treatment at the first water change on

day 3.  Extra organisms were haphazardly selected to be

discarded if there were not a sufficient number of empty test

wells available.

3 D21 Down sample spilled The 15ug/L (nominal) down sample reflecting day 21 test

solution was accidentally knocked over during the water

change process.

4 Temperature not recorded Temperature in the humidity chamber was not measured on

D1‐reading #2 and both readings on D2

5 Water chemistry values not

collected in test solutions

Dissolved Oxygen and pH were not collected in the Up and

Down test solutions.  Operators instead collected data from

dilution water prior to water change.  The basis for this

deviation is that operators interpreted the language of the

OECD 2009 guideline to indicate this was an optional

component (“should”, as opposed to “must” or “will”)  

6 Potential for evaporation data

not recorded.

A 14‐day dry run study was performed in June/July for the

operators to practice performing the test.  Salinity results on

water change days were reported to have been performed,

but the data was not recorded.

7 Lost copepods during pairing

process

Approximately 15 animals were lost in various plates spread

across the concentrations.

8 Mature male/female copepods

not immediately mated.  

Sexually mature copepods were scheduled to be paired for

mating on Day 18, a water change day.  D18 fell on a Saturday

and the laboratory workload coupled with reduced staff was

such that a water change and mating could not occur on the

same day.  BES elected to prioritize the water change and

delay the creation of mating pairs until D21.

9 Mating pairs not from same

microplate

In order to maximize the number of mating pairs, males and

females were paired across replicates but within treatment.  

However, we attempted to create male/female pairs within

replicate when possible.